TNFa may play a pathogenic role during HIV infection, especially at local tissue sites such as the lung. The newly described TNFa-converting enzyme (TACE) may play a central role in modulating TNFa activity, which in turn may be a major determinant of HIV replication. TACE activity may, however, differ markedly in lung as opposed to peripheral blood cells or lymph node tissues. Our preliminary data show that TNF activity in the lung, as determined by TNFa agonist and sTNFRII antagonist concentrations, decreases as HIV disease progresses, CD4 cell counts fall, and viral load increases. This stands in contrast to reports using plasma or peripheral blood mononuclear cells (PBMC), or that measure only agonist, but not antagonist, activity of TNFa in blood. Our central hypothesis is that decreased ERK signaling and increased TACE activity causes these changes in TNF activity with progressive HIV infection in lung cells of HIV+ subjects. We have preliminary data to support this central hypothesis. We will test whether these changes are caused by TACE effects on ERK, increased TACE through inducible nitric oxide synthase (iNOS) and PC-PLC pathways, through autocrine effects of TNFa, or by post-transcriptional effects of Tp12 on TNFa translation, and whether they are limited to particular cells in the lung. Patient-derived samples from carefully selected subjects studied pre- and post-highly-active antiretroviral therapy (HAART), examined in an ex vivo model of chronic HIV infection of alveolar macrophages (AMs), will be used in these experiments. Comparisons will be made with simultaneously obtained PBMC, and with AMs and PBMC from HIV-negative control donors. Changes in ERK and TACE activity will be correlated with HIV replication and TNFa on a true single cell level using quantitative image analysis in lung cells and tissues to determine whether these mechanisms are operative in vivo in A M s or other lungs cells. This hypothesis-driven project will suggest whether anti-TNF or anti-TACE therapies will improve immunologic function and virologic control in HIV-infected persons receiving HAART.